CN104137778A - Method for culturing bletilla striata protocorm by means of bletilla striata seeds according to fluid suspension culture method - Google Patents

Method for culturing bletilla striata protocorm by means of bletilla striata seeds according to fluid suspension culture method Download PDF

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CN104137778A
CN104137778A CN201410368387.6A CN201410368387A CN104137778A CN 104137778 A CN104137778 A CN 104137778A CN 201410368387 A CN201410368387 A CN 201410368387A CN 104137778 A CN104137778 A CN 104137778A
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bletilla
protocorm
bletilla striata
culture
cultivation
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CN201410368387.6A
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CN104137778B (en
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周义峰
汤兴利
刘彦红
吴亚萍
王玫
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JIANGSU PIPA LANDSCAPE CO Ltd
Nanjing Agricultural University
Dongtai Institute of Tidal Flat Research of CAS
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JIANGSU PIPA LANDSCAPE CO Ltd
Nanjing Agricultural University
Dongtai Institute of Tidal Flat Research of CAS
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Abstract

The invention discloses a method for culturing bletilla striata protocorm by means of bletilla striata seeds according to the fluid suspension culture method. The method for culturing the bletilla striata protocorm by means of the bletilla striata seeds according to the fluid suspension culture method comprises the following steps that (1) the bletilla striata seeds are sterilized; (2) suspension germination cultivation is conducted, wherein under the sterile condition, a rhizoma bletillae capsule shell is cut open through scissors, and the seeds fall into a liquid germination culture medium through shaking and are cultured for thirty days in a shaking mode; (3) suspension culture of strong seedlings is conducted, wherein the bletilla striata protocorm obtained by conducting suspension germination cultivation in the step (2) is transferred into a liquid strong seedling culture medium and is cultured for 30-45 days in a shaking mode. According to the method for culturing the bletilla striata protocorm by means of the bletilla striata seeds according to the fluid suspension culture method, the fluid suspension culture method is adopted, the bletilla striata protocorm can be obtained effectively, operation is easy, cost is low, and the culture cycle can be effectively shortened.

Description

A kind of bletilla seed liquid suspension is cultivated the method for protocorm
Technical field
The present invention relates to a kind of bletilla seed seedling-raising technology, be specifically related to a kind of method that adopts liquid suspension cultural method bletilla to be carried out to Protocorm.
Background technology
Bletilla ( bletilla striata(Thunb.) Rchb. f.) for the orchid family bletilla belongs to perennial root herbaceous plant, the pseudobulb hyoscine of meat plumpness; A raceme tool number flower, flower purple or pale red, flower type is larger, 5 centimetres of diameters, form grace, has very high medical value and ornamental value.
Bletilla is main by division propagation under nature, and reproduction coefficient is lower; Although can produce a large amount of seeds, seed is difficult for germinateing.Because bletilla reproduction rate is low, cannot adapt to the demand of large-scale planting.Mainly concentrate on for the research of bletilla breeding at present that seed asepsis is auxiliary grows seedlings and organize cultivation, this technology can obtain a large amount of seedlings at short notice, is approach fast and effectively on bletilla is produced.
Existing bletilla group training is all selected to solid culture medium, when inoculation, bletilla seed is shaken off to the surface at medium gently, but because bletilla seed is Powdered, very tiny, when operation, be difficult to ensure that each seed can fully contact medium or embryo base portion touches medium, cause growth conditions difference, cause emerging irregular, and switch over operation is loaded down with trivial details, workload is huge.
Summary of the invention
To the object of the invention is the defect existing in prior art in order solving, a kind of method of easy to operate, bletilla seed culture protocorm that cost is low to be provided.
In order to achieve the above object, the invention provides a kind of method of bletilla seed liquid suspension cultivation protocorm, the method comprises the following steps:
(1) bletilla seed disinfection;
(2) suspension is germinateed and is cultivated: under aseptic condition, cut off bletilla capsule shell with scissors, seed is shaken off in liquid germination medium, shake cultivation; Liquid germination medium adopts 1/2MS medium (adopt the macroelement concentration of material in MS medium to reduce by half, other reagent concentration is with MS medium); In liquid germination medium, contain NAA 0.5-1.0mgL -1, sucrose 30gL -1; Condition of culture is dark cultivation, cultivation temperature 24-26 DEG C, and incubation time is 30 days;
(3) suspension strong seedling culture: the bletilla protocorm obtaining after the cultivation of germinateing suspending through step (2) is transferred in liquid strong seedling culture base, shakes cultivation; Described liquid strong seedling culture base adopts 1/2MS medium; In liquid strong seedling culture base, contain NAA 1.0mgL -1, 6-BA 1.0mgL -1, sucrose 30gL -1; Condition of culture is illumination cultivation, and light application time is 16h/d, and intensity of illumination is 2000-2500lx, and cultivation temperature is 24-26 DEG C, and incubation time is 30-45 days.
Wherein, in step (2), in liquid germination medium, the preferred concentration of NAA is 0.5mgL -1.Incubation time preferably 30 days in step (3).
In step (1), bletilla seed disinfection is adopted to following methods: by clean bletilla capsule surface clean, wash 5-10min with washing powder, putting flowing water rinses well, use again 75% alcohol surface sterilization 5min, aseptic water washing is clean, then use 10% liquor natrii hypochloritis's soaking disinfection bletilla capsule, aseptic water washing 5-7 time.
And the bletilla protocorm obtaining after step (3) suspension strong seedling culture can be proceeded to and in breeding and seedling nursing with equipment equipment, carry out hardening or transfer and in solid culture medium, carry out culture of rootage.
The concrete steps that bletilla seed liquid suspension of the present invention is cultivated protocorm are as follows:
1. bletilla seed disinfection: by bletilla capsule surface wash clean, with washing powder washing 5 ~ 10min, put flowing water and rinse well, use again 75% alcohol surface sterilization 5min, aseptic water washing is clean, then uses 10% liquor natrii hypochloritis's soaking disinfection bletilla capsule, aseptic water washing 5 ~ 7 times.
2. medium preparation: preparation 1/2MS+0.5mgL -1nAA+30gL -1bletilla liquid germination medium and the 1/2MS+1.0mgL of sucrose -1nAA+1.0mgL -16-BA+30gL -1the bletilla liquid strong seedling culture base of sucrose, under hot conditions, sterilizing is cooling rear stand-by.
3. inoculation: under aseptic condition, cut off bletilla capsule shell with scissors, seed is shaken off at 1/2MS+0.5mgL -1nAA+30gL -1the bletilla liquid of sucrose germinates in training base.
4. suspending germinates cultivates: the bletilla liquid germination medium of having inoculated is placed in to shaking table, and concussion is cultivated.Condition of culture is dark culturing, and cultivation temperature is 25 ± 1 DEG C, cultivates 30 days.
5. suspension strong seedling culture: the bletilla protocorm of cultivating after germinateing 30 days is transferred in 1/2MS+1.0mgL by suspending -1nAA+1.0mgL -16-BA+30gL -1in the bletilla liquid strong seedling culture base of sucrose, carry out strong seedling culture, condition of culture is for cultivation temperature is (25 ± 1) DEG C, intensity of illumination 2000 ~ 2500lx, light application time 16hd -1.Concussion is cultivated, and incubation time is 30-45 days.
6. bletilla seed suspension strong seedling culture finishes, and can proceed to and in breeding and seedling nursing with equipment equipment, carries out hardening or transfer and in solid culture medium, carry out culture of rootage, for preparing under bletilla seedling.
The present invention has the following advantages compared to existing technology: the present invention adopts liquid suspension to cultivate the former solid culture of replacement, can effectively ensure that each seed fully contacts medium or embryo base portion touches medium, emerge neat, and switch over operation is simple.The present invention utilizes liquid suspension to cultivate, and can effectively obtain bletilla protocorm, simple to operate, cost is low and can effectively shorten the production cycle.
Brief description of the drawings
Fig. 1 is the germination of bletilla under different illumination conditions;
Fig. 2 is the impact that different culture media germinates on bletilla.
In Fig. 1, a is the germination of bletilla under dark condition, and b is the germination of bletilla under illumination condition;
In Fig. 2,1 for adopting the impact of culture medium prescription 1 on bletilla germination, and 2 is the impact that employing culture medium prescription 2 germinates on bletilla, and 3 is the impact of employing culture medium prescription 3 on bletilla germination, and 4 is the impact that adopts culture medium prescription 4 to germinate on bletilla.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
1 materials and methods
1.1 material
1.1.1 experiment with material be bletilla ( bletilla striata(Thunb.) Rchb. f.) ripe capsule then.(provided by teacher Li Linhua of Anhui, City of Wuhu in Anhui traditional Chinese medicine junior college, Fruit combines in bifurcation mountain, Nanling County, Wuhu city)
1.1.2 reagent, instrument and equipment 6-benzyl aminoadenine (6-Benzylaminopurine, be called for short 6-BA) purchased from Shanghai Wan Jiang Bioisystech Co., Ltd, 1-methyl α-naphthyl acetate (1-Naphthaleneacetic acid is called for short NAA) purchased from Chinasun Specialty Products Co., Ltd; The OptiMair of ESCO company superclean bench; Shenan Medical Appliances Factory, Shanghai's high-pressure sterilizing pot; Electronic balance is provided by Haikang, Shanghai Electronic Instruments Plant, and anatomical lens is Olympus CX41-12C02 binocular microscope CX41 Shanghai Ze Shi Electro-optical Technology, INC. (US) 62 Martin Road, Concord, Massachusetts 017; The inoculating tool such as scalpel, filter paper is all purchased from Nanjing Shou De experiment equipment Co., Ltd.Other chemical reagent is purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
1.2 method
1.2.1 material processed and inoculation bletilla striata capsule surface clean are clean, detergent immersion washing 5 ~ 10min, and flowing water is rinsed well, 75% alcohol submergence sterilization 5min, aseptic water washing is clean, 10% liquor natrii hypochloritis's soaking disinfection bletilla capsule, aseptic water washing 5 ~ 7 times.Under aseptic condition, sterile scissors is cut off bletilla capsule shell, and seed is evenly shaken off to liquid nutrient medium.
1.2.2 the preparation of liquid nutrient medium, taking 1/2MS liquid nutrient medium as basis, is added 30gL -1sucrose, regulate pH to 5.8, hormone NAA and 6-BA arrange 3 different concentration gradients (0,0.5mgL -1, 1.0mgL -1).Culture medium prescription is in table 1.
Table 1 bletilla germinates and protocorm generates medium allocation list
1.2.3 condition of culture is secretly cultivated cultivation temperature for (25 ± 1) DEG C, unglazed photograph.It is 25 ± 1 DEG C that light is cultivated cultivation temperature, intensity of illumination 2000-2500lx, light application time 16hd -1.Concussion is cultivated.
1.2.4 DATA REASONING and statistical analysis detect chitting piece average sample for examination, take pictures, measure and statistical data under anatomical lens.Length is the total length of protocorm and bud, and rugosity is protocorm thickness.Unit is millimeter (㎜).Every processing repeats 7 times, and carries out statistical analysis, and analysis software is SPSS variance analysis software.
2 results and analysis
The impact of 2.1 different illumination conditions on bletilla seed sprouting
Be placed under dark and illumination condition and cultivate being inoculated in after bletilla seed disinfection in No. 2 medium, cultivate 30 days, sample observation, statistical experiment data are as following table (table 2).Seed sprouting situation as shown in Figure 1.
Bletilla germination under table 2 different illumination conditions
Condition of culture Length (㎜) Rugosity (㎜) Length/rugosity Germination rate
Dark condition 0.993±0.202** 0.350±0.053* 2.942±0.797** 98%±2%
Illumination condition 0.336±0.053* 0.216±0.015* 1.565±0.301* 75%±3%
(* * represents extremely significantly P < 0.01, and * represents remarkable P < 0.05)
Experimental result demonstration, dark condition is more conducive to bletilla seed sprouting, and bletilla germination rate approximately 80% under optical condition, than low 20% left and right of dark condition.Meanwhile, formation and the early growth of illumination to bletilla protocorm also has obvious inhibitory action.Under optical condition, the length of bletilla protocorm, rugosity are only all 30% and 60% left and right of dark condition, exist extremely significantly and significant difference.Therefore, can infer, under exogenous hormone and well-fed condition, although optical condition is not the decisive factor of bletilla germination and early growth, but can produce material impact to its formation and growth, dark condition can promote bletilla seed sprouting, protocorm to form and growth.
Experiment is discovery simultaneously, and appropriate illumination meeting makes bletilla protocorm present green, should, to its late growing stage, particularly in the environment of exogenous hormone and the supply of exogenous nutrition material, have positive meaning.
The impact of 2.2 different culture medias on bletilla seed sprouting
Be placed under dark condition and cultivate 30 days being inoculated in after bletilla seed disinfection in different culture media, sample observation, statistical experiment data are as following table (table 3).Seed sprouting situation as shown in Figure 2.
The impact that table 3 different culture media germinates on bletilla
Culture medium prescription Length (㎜) Rugosity (㎜) Length/rugosity Seed germination rate
1 1.209±0.072** 0.230±0.018* 5.295±0.667** 85%±5%
2 0.993±0.202** 0.350±0.053** 2.942±0.797 98%±2%
3 0.297±0.060* 0.179±0.043* 1.792±0.665 30%±2%
4 0.297±0.037* 0.049±0.008** 6.131±1.250** 25%±5%
(* * represents extremely significantly P < 0.01, and * represents remarkable P < 0.05)
Upper table data show, different culture media formula forms and all has considerable influence bletilla seed germination rate and protocorm.The 6-BA adding in formula 3 and formula 4 obviously suppresses bletilla and germinates, and to bletilla protocorm, growth has obvious inhibitory action.Therefore in bletilla seed sprouting process, should not add 6-BA.Although formula 1 and 2 can promote the elongation of bletilla bud, on bletilla protocorm, impact is mainly manifested in the elongation growth that promotes bletilla bud to formula 1, and excessive growth phenomenon appears in seedling, and protocorm increases not obvious.And bletilla percentage of seedgermination is high in formula 2, protocorm growth rate is consistent, is the formula of comparatively ideal bletilla seed sprouting.The optimum formula of therefore advising bletilla seed sprouting liquid nutrient medium is 1/2MS+0.5mgL -1nAA+30gL -1sucrose.NAA concentration range can be extended to 1. 0mgL -1, but should not be higher, can there is the excessive growth of bletilla seedling.
2.3 different culture medias are on the bletilla impact in strong sprout
By being inoculated in No. 2 medium and cultivating bletilla protocorm after 30 days and transfer and cultivate under dark condition, cultivate after 30 days sampling and measuring statistics under illumination condition in different culture media.Experimental result is as follows.
Table 4 different culture media is on the bletilla impact in strong sprout
Culture medium prescription Length (㎜) Rugosity (㎜) Increase (㎜) Increase thick (㎜)
1 1.765±0.167** 0.456±0.067* 0.772 0.106
2 1.041±0.075 0.418±0.059 0.048 0.068
3 1.664±0.167** 0.434±0.038* 0.671 0.084
4 1.696±0.170** 0.512±0.083** 0.703 0.162
5 1.218±0.135 0.363±0.072 0.225 0.013
6 1.164±0.083 0.359±0.059 0.171 0.015
7 1.384±0.174* 0.465±0.055* 0.391 0.115
8 1.415±0.131* 0.406±0.121 0.422 0.056
(* * represents extremely significantly P < 0.01, and * represents remarkable P < 0.05)
Upper table data demonstration, NAA concentration is 0 o'clock, bletilla protocorm increases slightly not obvious, and seedling increases slightly also not obvious; When NAA concentration is 0.5mgL -1time bletilla protocorm increase slightly not obvious, obviously thick but seedling increases.Illustrate that the NAA of low concentration can not control the growth rate of bletilla well, can not reach the object in strong sprout.From upper table, can obviously find out and adopt culture medium prescription 4 to have obvious facilitation to the rugosity increase of bletilla, the length increase of bletilla is also comparatively obvious, can Fast Growth, reach the object in strong sprout.Therefore the strong seedling culture based formulas of bletilla is with 1/2MS+1.0mgL -1nAA+1mgL -16-BA+30gL -1sucrose is good.
In sum, the best of bletilla germination and protocorm breeding condition are for to be inoculated in 1/2MS+30gL by bletilla seed -1sucrose+0.5mgL -1in the liquid nutrient medium of NAA, cultivation temperature is 25 ± 1 DEG C, and concussion is dark cultivated after 30 days, transferred into 1/2MS+30gL -1sucrose+1.0mgL -1nAA+1.0mgL -1in the liquid nutrient medium of 6-BA, carry out strong seedling culture, condition of culture is that cultivation temperature is (25 ± 1) DEG C, intensity of illumination 2000-2500lx, light application time 16hd -1.Concussion is cultivated.Incubation time is 30-45 days.

Claims (5)

1. bletilla seed liquid suspension is cultivated a method for protocorm, it is characterized in that, comprises the following steps:
(1) bletilla seed disinfection;
(2) suspension is germinateed and is cultivated: under aseptic condition, cut off bletilla capsule shell with scissors, seed is shaken off in liquid germination medium, shake cultivation; Described liquid germination medium adopts 1/2MS medium; In described liquid germination medium, contain NAA 0.5-1.0mg/L, sucrose 30g/L; Described condition of culture is dark cultivation, cultivation temperature 24-26 DEG C, and incubation time is 30 days;
(3) suspension strong seedling culture: the bletilla protocorm obtaining after the cultivation of germinateing suspending through step (2) is transferred in liquid strong seedling culture base, shakes cultivation; Described liquid strong seedling culture base adopts 1/2MS medium; In described liquid strong seedling culture base, contain NAA 1.0mg/L, 6-BA 1.0mg/L, sucrose 30g/L; Described condition of culture is illumination cultivation, and light application time is 16h/d, and intensity of illumination is 2000-2500lx, and cultivation temperature is 24-26 DEG C, and incubation time is 30-45 days.
2. method according to claim 1, is characterized in that, in described step (2), in liquid germination medium, the content of NAA is 0.5mg/L.
3. method according to claim 1, is characterized in that, in described step (3), incubation time is 30 days.
4. according to the arbitrary described method of claims 1 to 3, it is characterized in that, in described step (1), bletilla seed disinfection is adopted to following methods: by clean bletilla capsule surface clean, wash 5-10min with washing powder, put flowing water and rinse well, then use 75% alcohol surface sterilization 5min, aseptic water washing is clean, then use 10% liquor natrii hypochloritis's soaking disinfection bletilla capsule, aseptic water washing 5-7 time.
5. according to the arbitrary described method of claims 1 to 3, it is characterized in that, the bletilla protocorm obtaining after described step (3) suspension strong seedling culture proceeds to and in breeding and seedling nursing with equipment equipment, carries out hardening or transfer and in solid culture medium, carry out culture of rootage.
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CN104737911A (en) * 2015-03-30 2015-07-01 云南中医学院 Quick cultivation method for rhizoma bletillae tissue culture seedlings
CN105075858A (en) * 2015-05-29 2015-11-25 浙江中医药大学 Liquid rapid-propagation method for rhizoma bletillae seeds
CN105325302A (en) * 2015-12-11 2016-02-17 电子科技大学 Method for bletilla striata seedling production based on liquid medium
CN105340740A (en) * 2015-11-10 2016-02-24 云南师范大学 Liquid high-efficiency germination induction solution and rapid propagation method for bletilla striata seeds
CN105766645A (en) * 2016-03-31 2016-07-20 广西医科大学 Efficient reproduction method of rhizoma bletillae tissue culture seedlings
CN106478253A (en) * 2016-10-14 2017-03-08 贵州万重山生态农业开发有限公司 A kind of two-part Pseudobulbus Bletillae (Rhizoma Bletillae) direct sowing and seedling method
CN106857250A (en) * 2017-01-22 2017-06-20 安徽振扬农林生态开发有限公司 A kind of method for promoting bletilla striata seeds to sprout
CN110073979A (en) * 2019-06-04 2019-08-02 红河学院 A kind of method for culturing seedlings of orchid
CN110547194A (en) * 2019-09-12 2019-12-10 浙江省东阳玉米研究所 Tissue culture and rapid propagation method for seeds of bletilla striata
CN112616657A (en) * 2020-12-02 2021-04-09 云南省农业科学院农业环境资源研究所 Sterile preservation method of bletilla striata seeds

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Publication number Priority date Publication date Assignee Title
CN104737911A (en) * 2015-03-30 2015-07-01 云南中医学院 Quick cultivation method for rhizoma bletillae tissue culture seedlings
CN105075858A (en) * 2015-05-29 2015-11-25 浙江中医药大学 Liquid rapid-propagation method for rhizoma bletillae seeds
CN105340740A (en) * 2015-11-10 2016-02-24 云南师范大学 Liquid high-efficiency germination induction solution and rapid propagation method for bletilla striata seeds
CN105325302A (en) * 2015-12-11 2016-02-17 电子科技大学 Method for bletilla striata seedling production based on liquid medium
CN105766645A (en) * 2016-03-31 2016-07-20 广西医科大学 Efficient reproduction method of rhizoma bletillae tissue culture seedlings
CN105766645B (en) * 2016-03-31 2018-07-24 广西医科大学 Bletilla tissue-cultured seedling efficient propagation method
CN106478253A (en) * 2016-10-14 2017-03-08 贵州万重山生态农业开发有限公司 A kind of two-part Pseudobulbus Bletillae (Rhizoma Bletillae) direct sowing and seedling method
CN106857250A (en) * 2017-01-22 2017-06-20 安徽振扬农林生态开发有限公司 A kind of method for promoting bletilla striata seeds to sprout
CN110073979A (en) * 2019-06-04 2019-08-02 红河学院 A kind of method for culturing seedlings of orchid
CN110547194A (en) * 2019-09-12 2019-12-10 浙江省东阳玉米研究所 Tissue culture and rapid propagation method for seeds of bletilla striata
CN112616657A (en) * 2020-12-02 2021-04-09 云南省农业科学院农业环境资源研究所 Sterile preservation method of bletilla striata seeds
CN112616657B (en) * 2020-12-02 2022-01-07 云南省农业科学院农业环境资源研究所 Sterile preservation method of bletilla striata seeds

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